Anne W. Scribner

Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity

Hyperaldosteronism is associated with impaired vascular reactivity; however, the mechanisms by which aldosterone promotes endothelial dysfunction remain unknown. Glucose-6-phosphate dehydrogenase (G6PD) modulates vascular function by limiting oxidant stress to preserve bioavailable nitric oxide (NO•). Here we show that aldosterone (10−9–;10−7 mol/l) decreased endothelial G6PD expression and activity in vitro, resulting in increased oxidant stress and decreased NO• levels—similar to what is observed in G6PD-deficient endothelial cells. Aldosterone decreased G6PD expression by increasing expression of the cyclic AMP−response element modulator (CREM) to inhibit cyclic AMP−response element binding protein (CREB)-mediated G6PD transcription. In vivo, infusion of aldosterone decreased vascular G6PD expression and impaired vascular reactivity. These effects were abrogated by spironolactone or vascular gene transfer of G6pd. These findings demonstrate that aldosterone induces a G6PD-deficient phenotype to impair endothelial function; aldosterone antagonism or gene transfer of G6pd improves vascular reactivity by restoring G6PD activity.

Glucose-6-Phosphate Dehydrogenase Overexpression Decreases Endothelial Cell Oxidant Stress and Increases Bioavailable Nitric Oxide

Objective—Glucose-6-phosphate dehydrogenase (G6PD), the principal source of NADPH, serves as an antioxidant enzyme to modulate the redox milieu and nitric oxide synthase activity. Deficient G6PD activity is associated with increased endothelial cell oxidant stress and diminished bioavailable nitric oxide (NO·). Therefore, we examined whether overexpression of G6PD would decrease reactive oxygen species accumulation and increase bioavailable NO· in endothelial cells. Methods and Results—Adenoviral-mediated gene transfer of G6PD increased G6PD expression, activity, and NADPH levels in bovine aortic endothelial cells (BAECs). BAECs overexpressing G6PD demonstrated a significant reduction in reactive oxygen species accumulation when exposed to hydrogen peroxide, xanthine-xanthine oxidase, or tumor necrosis factor-&agr; compared with BAECs with basal levels of G6PD. BAECs overexpressing G6PD maintained intracellular glutathione stores when exposed to oxidants because of increased activity of glutathione reductase, an effect that was not observed in endothelial cells with normal G6PD activity. Overexpression of G6PD was also associated with enhanced nitric oxide synthase activity, resulting in elevated levels of cGMP, nitrate, and nitrite, and this response was increased after stimulation with bradykinin. Conclusions—Overexpression of G6PD in vascular endothelial cells decreases reactive oxygen species accumulation in response to exogenous and endogenous oxidant stress and improves levels of bioavailable NO·.

Glucose-6-phosphate dehydrogenase deficiency promotes endothelial oxidant stress and decreases endothelial nitric oxide bioavailability

The vascular endothelium compensates for oxidant stress by increasing the activity of antioxidant enzymes such as glucose‐6‐phophate dehydrogenase (G6PD). G6PD provides reducing equivalents of NAPDH to maintain glutathione stores and modulates nitric oxide synthase (eNOS) activity. To determine whether deficient G6PD activity perturbs these responses, we treated bovine aortic endothelial cells with dehydroepiandrosterone or an antisense oligodeoxynucleotide to G6PD mRNA to decrease G6PD activity and expression. When exposed to hydrogen peroxide, reactive oxygen species (ROS) accumulation was increased in G6PD‐deficient cells compared with those with normal activity. To determine the source of increased oxidant stress in G6PD‐deficient cells, we used inhibitors of ROS generation, which suggested that eNOS was contributing to ROS production. Treatment with L‐NMMA, an inhibitor of eNOS mediated‐nitric oxide (NO) but not superoxide, production confirmed this observation; in contrast to L‐NAME, L‐NMMA promoted ROS generation in G6PD‐deficient cells. In addition, deficient G6PD activity was associated with a decrease in endothelium‐derived bioavailable NO in response to the agonists A23187 and bradykinin as demonstrated by decreased endothelial cGMP and nitrate/nitrite levels. Enhanced ROS accumulation and decreased NO bioavailability may represent one mechanism by which G6PD deficiency contributes to vascular oxidant stress and endothelial dysfunction.

Salt-induced hypertension in Dahl salt-resistant and salt-sensitive rats with NOS II inhibition.

Although recent evidence suggests that reduced nitric oxide (NO) production may be involved in salt-induced hypertension, the specific NO synthase (NOS) responsible for the conveyance of salt sensitivity remains unknown. To determine the role of inducible NOS (NOS II) in salt-induced hypertension, we treated Dahl salt-resistant (DR) rats with the selective NOS II inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) for 12 days. Tail-cuff systolic blood pressures rose 29 +/- 6 and 42 +/- 8 mmHg in DR rats given 150 and 300 nmol AMT/h, respectively (P < 0.01, 2-way ANOVA) after 7 days of 8% NaCl diet. We observed similar results with two other potent selective NOS II inhibitors, S-ethylisourea (EIT) and N-[3-(aminomethyl)benzyl]acetamidine hydrochloride (1400W). Additionally, AMT effects were independent of alterations in endothelial function as assessed by diameter change of mesenteric arterioles in response to methacholine using videomicroscopy. We, therefore, conclude from these data that NOS II is important in salt-induced hypertension.

Prevention of hypertension and renal dysfunction in Dahl rats by α-tocopherol

&NA; Although hypertension is a risk factor for the development of end‐stage renal disease, not all hypertensive patients progress to develop renal dysfunction. The mechanisms underlying hypertensive nephropathy are poorly understood. The authors have recently shown that the development of hypertension and renal dysfunction is accompanied by an accumulation of partially reduced oxygen and its derivatives, known collectively as reactive oxygen species. In the present study, the effect of a lipid‐soluble antioxidant on the development of salt‐dependent hypertensive nephropathy was evaluated in the Dahl rat. It was found that a high‐salt diet (8% NaCl) led to the development of hypertension, increased renal oxidative stress (superoxide production and 8‐epi‐prostaglandin F2&agr;), and decreased glomerular filtration rate and renal plasma flow in the Dahl salt‐sensitive (DSS) rat, and that these adverse effects of salt were prevented by supplementing the high‐salt diet with 1000 U/kg chow of &agr;‐tocopherol. It is well known that urinary cyclic guanosine monophosphate (cGMP) levels are lower in hypertensive DSS rats than in Dahl salt‐resistant (DSR) rats on a high‐salt diet. Most surprisingly, when supplemented with &agr;‐tocopherol, DSS rats on an 8% NaCl diet were able to excrete as much cGMP as DSR rats. Taken together, these findings suggest that, in the DSS rat, salt‐dependent hypertensive nephropathy and decreased nitric oxide bioavailability are associated with increased oxidative stress, and that antioxidants can preclude these adverse effects of salt feeding, and consequently, prevent salt‐dependent hypertension and nephropathy.